Diabetes mellitus represents a collection of genetically determined disorders one of which is the altered metabolism of lipids associated with a deficiency of insulin or insulin resistance. Diabetics are prone to certain long-term complications. The range of medical problems includes cardiovascular disease, retinopathy, nephropathy, and neuropathy. The latter condition is a neuropathological disorder of the peripheral nervous system, which leads to a reduction in nerve conduction velocity in both motor and sensory nerves. The pathophysiology of diabetic peripheral neuropathy could be associated with the abnormal metabolism of essential fatty acids (Julu P., 1998, in Essential Fatty Acids and Eicosanoids, AOCS Press, Ill., U.S.A.,pp. 168-175). This abnormal or altered lipid metabolism is reflected in the lack of incorporation of n-6 fatty acids in membrane phospholipids (Coste et al., 1999, J. Nutr. Biochem., 10: 411-420). Evidence from experimental diabetes studies in animals indicates that the biosynthesis of polyunsaturated fatty acids (PUFAs) by the desaturation and elongation systems is impaired. A multi-center clinical trial supported by Scotia Pharmaceuticals (UK) has demonstrated that providing n-6 fatty acids, such as gamma-linolenic acid (GLA), to patients with mild diabetic neuropathy alleviates the course of the disease (Keen et al., 1993, Diabetes Care, 16: 8-15).
PUFAs are also known to cause cell cycle arrest, induction of apoptosis, inhibition of mitosis (Seegers et al., 1997, Prostaglandins Leukot. Essent. Fatty Acids, 56: 271-280 and Lai et al., 1996, Br. J. Cancer, 74: 1375-1383) and cell proliferation (Calviello et al., 1998, Int. J. Cancer, 75: 699-705), reduction of tumor-endothelium adhesion, improvement of gap junction communications of the endothelium (Jiang et al., 1997, Prostaglandins Leukot. Essent Fatty Acids, 56: 307-316), inhibition of urokinases (du Toit et al., 1994, Prostaglandins Leukot. Essent. Fatty Acids, 51: 121-124) reduction of the effects of growth factors on cancer cells (Jiang et al., 1995b, Br. J. Cancer, 71: 744-752), reversion of multi-drug resistance (Weber et al., 1994, J. Nat Cancer Inst., 86: 638-639), and increase of the cytotoxic effects of chemotherapeutic agents (Plumb et al., 1993, Br. J. Cancer, 67: 728-733 and Anderson et al., 1998, Anticancer Res., 18: 791-800).
N-3 and n-6 fatty acids follow a similar route of metabolism and it is likely that individual enzymes act on similar substrates in both PUFA families. In the n-6 pathway dihomogammalinolenic acid (DGLA, 20:3n-6) is converted to arachidonic acid (AA, 20:4n-6) through desaturation by an enzyme known as delta-5-desaturase (D5D). This enzyme also produces eicosapentaenoic acid (EPA, 20:5n-3) from delta 8, 11, 14, 17 eicosatetraenoic (20:4n-3) in the n-3 pathway.
Delta-5-desaturase belongs to a subclass of enzymes known as “front-end” desaturases, which introduce double bonds into the acyl chain between the carboxyl group and an existing double bond. Delta-5-desaturase is an enzyme bound to the membrane of the endoplasmic reticulum, which is part of a system that consists of three major proteins associated with an electron transport chain (Fujiwara et al., 1983, Biochem. Biophys. Res. Commmun, 110: 36-41 and 1984, Arch. Biochem. Biophys., 233: 402-407). The cloning, expression and fatty acid regulation of the human delta-5-desaturase has been previously described (Cho et al., 1999b, J. Biol. Chem., 274: 37335-37339; Leonard et al., 2000, Biochem. J., 347: 719-724 and Marquardt et al., 2000, Genomics, 66: 175-183).
Both arachidonic acid (AA) and eicosapentaenoic acid (EPA), direct products of delta-5-desaturase, have been proposed as key PUFAs associated with a variety of diseases. A chronic cellular imbalance between AA and EPA and their respective eicosanoid derivatives may have major health implications. This imbalance has been implicated in arterial hypertension, hypercholesterolemia, atherosclerotic heart disease, chronic inflammatory and autoimmune disorders, allergic eczema and other atopic disorders (Booyens et al., 1985, Med. Hypotheses, 18: 53-60). In this regard, the fatty acid analysis of a group of the plasma phospholipid fraction of patients with coronary heart disease revealed that the levels of AA, EPA, gamma-linolenic acid (GLA), and docosahexaenoic acid (DHA) are low. In patients with essential hypertension, linoleic acid (LA) and AA are also low. (Das U., 1995, Prostaglandins Leukot. Essent Fatty Acids, 52: 387-391). Furthermore, bioavailability of eicosanoid precursors, and in particular of AA, could affect several vascular functions and have a bearing on the pathogenesis or complications of hypertension (Russo et al., 1997, Hypertension, 4: 1058-1063). It has been also shown that the percentages of AA-containing species of phosphatidylcholine (PC) were lowered in the plasma of patients with hypercholesterolemia. The same PC species with AA were decreased in the patient's platelets (Dobner P, and Engelmann B., 1998, Am. J. Physiol., 275: E777-E784).
In addition to serving as the precursor to eicosanoids and other bioactive molecules, AA may function as a second messenger (Khan et al., 1995, Cell. Signal., 7: 171-184).
The proinflammatory eicosanoids prostaglandin E2 and leukotriene B4 are derived from AA, which is maintained at high cellular concentrations by the high n-6 and low n-3 polyunsaturated fatty acid content of the modern Western diet (James et al., 2000, Am. J. Clin. Nutr., 71: 343S-348S). Recent results have suggested that by inhibiting delta-5-desaturation, the production of prostaglandin E2 can be reduced with a concomitant reduction of inflammatory processes such as rheumatoid arthritis (Chavali S. R. and Forse R. A. 1999, Prostaglandins Leukot Essent. Fatty Acids, 61: 347-352; Navarro et al, 2000, J. Rheumatol., 27: 298-303). Indeed, AA-derived eicosanoid are pro-inflammatory and regulate the functions of cells of the immune system. Consumption of fish oils leads to replacement of AA in cell membranes by EPA. This changes the amount and alters the balance of eicosanoids produced. Consumption of oils rich in EPA diminishes lymphocyte proliferation, T-cell-mediated cytotoxicity, natural killer cell activity, macrophage-mediated cytotoxicity, monocyte and neutrophil chemotaxis, major histocompatibility class II expression and antigen presentation, production of pro-inflammatory cytokines (interleukins 1 and 6, tumour necrosis factor) and adhesion molecule expression. (Calder P. C., 1998, Braz. J. Med. Biol. Res. 4: 467-490). In experimentally induced T-cell-mediated autoimmune disease, essential fatty acid-deficient diets or diets supplemented with n-3 fatty acids appear to augment disease, whereas n-6 fatty acids prevent or reduce the severity. The regulation of gene expression, signal transduction pathways, production of eicosanoids and cytokines, and the action of antioxidant enzymes are all mechanisms by which dietary n-6 and n-3 fatty acids may exert effects on the immune system and autoimmune disease. (Harbige L. S., 1998, Proc. Nutr. Soc., 4: 555-562).
Patients with atopic dermatitis (eczema) have reduced levels of delta-5-desaturase products such as AA (Hansen A. E., 1933, Proc. Soc. Exp. Biol. Med., 31: 1160-1161). These findings are consistent with the other studies in which eczema patients had low levels of serum AA (Manku et al., 1984, Br. J. Dermatol., 110: 643-680). The therapeutic usefulness of n-6 PUFA supplementation in atopic eczema has been reported (Wright S. and Burton J. L., 1982, Lancet, 2: 1120-1122). These treatments are not restricted to n-6 PUFAs since beneficial effects have also been obtained using an ointment containing EPA and DHA (Watanabe T. and Kuroda Y., 1999, J. Med. Invest., 46: 173-177).
Psoriasis is another disease associated with. It is a multi-factorial skin disease characterized by a profound increase of free AA and its proinflammatory metabolites. Providing an alternative precursor (i.e. EPA) which competes with AA in the eicosanoid synthesis, the prominflammatory effect is reduced by inducing the production of less inflammatory and chemotactic derivatives with a concomitant reduction of the chronic plaque-type psoriasis (Mayser et al., 1998, J. Am. Acad. Dermatol. 38: 539-547).
Similarly, acute respiratory distress syndrome (ARDS) characterized by acute lung injury, is linked with excessive release of AA-derived inflammatory mediators and toxic oxygen radicals from activated intrapulmonary macrophages and neutrophils. Supplementation of nutritional formulas with combination of EPA and GLA which favors an anti-inflammatory state, is used as adjuvant therapy in the clinical management of patients with or at risk of developing ARDS (Gadek et al., 1999, Crit. Care Med. 27: 1409-1420).
There is also an association of lipid accumulation in general and AA, in particular, with histological severity of human joint pathology (Lippiello et al., 1991, Metabolism 40: 571-576). Recent findings demonstrate that n-3 fatty acid supplementation affects molecular mechanisms that regulate the expression of catabolic factors involved in articular cartilage degradation (ACD). This shows the beneficial role of n-3 PUFAs in mitigating the physiological parameters that cause and propagate arthritic disease (Curtis et al., 2000, J. Biol. Chem. 275: 721-724).
AA and EPA have varying effects on cancer in mammalian cells. Studies have shown that n-3 and n-6 PUFAs and/or their metabolites are able to modulate the expression of tumor suppressors (Jiang et al., 2000, Prostaglandins Leukot. Essent Fatty Acids, 62: 119-127), lead to anti-metastatic mechanisms (Jiang et al., 1998a, Br. J. Cancer, 77: 731-738 and 1998b, Biochem. Biophys. Res. Commun., 244: 414-420) and modulate the expression of cell adhesion molecules including E-cadherin, desmoglein and beta-catenin (Jiang et al., 1995, Cancer Res., 55: 5043-5048 and Jiang et al., 2000, Prostaglandins Leukot. Essent. Fatty Acids, 62: 119-127).
In particular, EPA has been reported to significantly inhibit the growth of human pancreatic cancer cell lines in vitro (Falconer et al., 1994, Br. J. Cancer, 69: 826-832) and down-regulate the acute-phase response in patients with pancreatic cancer cachexia (Wigmore et al., 1997, Clin. Sci., 92: 215-221). It has also been shown that following exposure to EPA, malignant cells generate much higher levels of potentially cytotoxic superoxide radicals and lipid peroxidation products (Takeda et al., 1993, Anticancer Res., 13: 193-199).
Fungi, microalgae and rat liver microsomes have been used in different laboratories to test inhibitors of fatty acid delta-5-desaturase (Kawashima et al., 1996, Biosci Biotech. Biochem., 60: 1672-1676 and Obukowicz et al., 1998, Biochem. Pharmacol., 55: 1045-1058). However, these models which use different species are limited by the fact that they are not close enough to the desired target, i.e. the human delta-5-desaturase, for drug screening. Hence, it is desirable to develop an improved model and methodology for identifying-agents that modulate the activity of mammalian and, in particular, human fatty acid desaturases. The use of human delta-5-desaturase in whole cells, spheroplasts, and microsomes or as the purified enzyme from transformed yeast that overexpress the desaturase is a practical approach to test chemical libraries for modulators of the enzyme. The transformed yeast model also eliminates potential ethical concerns that may arise when human or mammalian tissues are used to obtain large amount of these enzymes for drug screening.
In this regard, an experimental model that can be manipulated to study the expression of genetic material isolated from humans and other species is needed to establish the role and function of these genes and their corresponding proteins in PUFA metabolism. This is particularly so in recognition of the fact that the relationship between a protein's unique role in a metabolic pathway and the expression of the gene encoding that protein is normally a well coordinated event such that subtle deviations can often lead to abnormal physiological processes. Moreover, such a system would facilitate the discovery and identification of candidate drug targets, which act at the DNA or protein level in order to correct abnormalities or imbalances in lipid metabolic changes associated with certain pathological conditions, such as diabetic neuropathy.
Towards developing such a system, it is critical to locate the control region for the delta-5-desaturase gene.